Lentiviral shRNA-Mediated CYBA Knockdown

CYBA (gene encoding p22phox) knockdown was performed by lentiviral transduction of shRNA as previously described.^{31 (link), 32 (link)} Briefly, lentiviral shRNA constructs in the pLKO.1 Puro vector (Open Biosystems, Thermo Fisher Scientific) were ordered. Targets (sense) used for these studies were scrambled control (Ctrl) 5’- GTCTCCGAACGTGT CACGTT-3’ and CYBA (TRCN0000064581) 5’-CGCTTCACCCAGTGGTACTTT-3’. 3.5×10⁶ HEK293T cells, which were used to generate viral particles, were plated on a tissue culture treated 10cm dish the day before transfection. One 10cm plate was transfected per shRNA construct by the calcium phosphate method using the following constructs: 6 μg control or CYBA shRNA construct, 0.6 μg vesicular stomatitis virus (VSV-G), and 5.4 μg cytomegalovirus (CMV 8.9.1). Supernatant was collected after 72 hr. and centrifuged to remove debris. 1×10⁶ PLB-985 were mixed with the viral supernatant in the presence of 4 μg/ml polybrene in a 6 well plate and centrifuged at 1000× g in a swinging bucket centrifuge for 30 minutes at 32°C. Stable lines were created by selection of transduced cells with 1 μg/ml puromycin.